Shiga Toxin & Ricin: Mechanisms of Ribotoxic Stress

描述（由申请人提供）：蓖麻毒素和志贺毒素具有独特的作用模式，对宿主核糖体RNA造成不可逆的损伤。我们对这些生物恐怖制剂如何导致组织损伤的理解是不完整的，也没有药物可以预防或治疗这些制剂的中毒。志贺毒素或蓖麻毒素介导的核糖体损伤导致蛋白质合成的抑制以及宿主细胞信号转导的深刻变化。这种现象被称为“核素毒性应激反应”，人们对其了解甚少。这一建议的重点是了解：1)是什么引发了核素毒性应激反应；2)涉及哪些信号转导通路；3)可能导致疾病发病的下游效应是什么。我们的初步数据表明，蓖麻毒素和志贺毒素激活宿主应激激活蛋白激酶（SAPK）和丝裂原激活蛋白激酶（MAPK）途径。我们假设这些途径的激活有助于在这些药物中毒后观察到的肠道和其他组织损伤。蓖麻毒素和志贺毒素对这些途径的影响将通过培养肠上皮细胞和口服中毒幼兔模型进行研究。这些信号转导通路被刺激的机制将使用志贺毒素突变体和分离的志贺毒素亚单位进行评估。蓖麻毒素和志贺毒素对宿主细胞信号转导的直接影响将通过磷酸化特异性抗体和免疫沉淀激酶测定来研究。转染显性阴性构建体、RNA干扰和信号转导途径的特定化学抑制剂将用于剖析蓖麻毒素和志贺毒素改变细胞生理的机制。我们的研究表明，口服纯化志贺毒素2可诱导肠道损伤。在幼兔模型中，将测试sapkinase阻断化合物对预防口服志贺毒素或蓖麻毒素引起的肠道损伤的作用。这项工作将为研究核素毒性应激反应提供新的见解。了解志贺毒素和蓖麻毒素如何对宿主细胞造成损害，将揭示潜在的治疗靶点。这些发现将与意外或故意的蓖麻毒素或志贺毒素中毒有关，并可能证明可推广到受这些毒素影响的其他宿主靶器官。

DESCRIPTION (provided by applicant): Ricin and Shiga toxin share a unique mode of action, causing irreversible damage to host ribosomal RNA. Our understanding of how these bioterror agents cause tissue damage is incomplete and no medications exist to prevent or treat intoxication with these agents. Shiga toxin- or ricin-mediated ribosomal damage results in inhibition of protein synthesis as well as profound changes in host cell signal transduction. This phenomenon, called the "ribotoxic stress response", is poorly understood. This proposal is focused on understanding: 1) what initiates the ribotoxic stress response; 2) which signal transduction pathways are involved; and 3) what are the downstream effects that may contribute to disease pathogenesis. Our preliminary data indicate that ricin and Shiga toxin activate host stress activated protein kinase (SAPK) and mitogen activated protein kinase (MAPK) pathways. We hypothesize that activation of these pathways contributes to intestinal and other tissue damage observed following intoxication with these agents. Effects of ricin and Shiga toxins on these pathways will be studied using cultured intestinal epithelial cells and an infant rabbit model of oral intoxication. The mechanism(s) by which these signal transduction pathways are stimulated will be assessed using Shiga toxin mutants and isolated Shiga toxin subunits. Direct effects of ricin and Shiga toxin on host cell signal transduction will be studied using phosphorylation-specific antibodies and immunoprecipitation kinase assays. Transfection of dominant negative constructs, RNA interference, and specific chemical inhibitors of signal transduction pathways will be used to dissect the mechanisms by which ricin and Shiga toxin alter cell physiology. Our work has revealed that oral administration of purified Shiga toxin 2 induces gut damage. In the infant rabbit model, SAPKinase-blocking compounds will be tested for their effect on preventing the gut damage that occurs following oral administration of Shiga toxin or ricin. This work will provide new insight into the ribotoxic stress response. Understanding how Shiga toxin and ricin cause damage to host cells should reveal potential therapeutic targets. These findings will be relevant to either accidental or intentional intoxication with ricin or Shiga toxin, and may prove generalizable to other host target organs affected by these toxins.